PROJECT SUMMARY Understanding the role of human MrgX1 in pain is a critical step to developing new therapy for pain treatment. However, the function of MrgX1 cannot be fully inferred from studying of its rodent ortholog MrgC. The cross-species variation in Mrg receptor function and agonist activity has presented a daunting challenge for the study of MrgX1 in animal models. To solve these problems, we have generated novel MrgX1:Mrg-/- mice that selectively express MrgX1 in DRG neurons, developed MrgX1-specific agonists and positive allosteric modulators (PAMs), and acquired a human neuronal HC-1 cell line. Using these new tools, we will examine the roles and mechanisms of MrgX1 in pain modulation and morphine analgesia for the first time. In Aim 1, we will determine the effects of MrgX1 agonists and PAMs on neuropathic pain-related behavior in MrgX1:Mrg-/- mice. We will characterize the analgesic properties of MrgX1 agonists (BAM8-22, JHU23) and two leading PAMs (891 and 179) in MrgX1:Mrg-/- mice after nerve injury. We will further examine if PAMs potentiate MrgX1 agonist-induced pain inhibition. Aim 1 will provide proof of principle for the therapeutic utility of using MrgX1 agonists and PAMs to treat neuropathic pain. In Aim 2, we will examine novel mechanisms of pain inhibition by MrgX1 agonists and PAMs in MrgX1:Mrg-/- mice and human neuronal HC-1 cells. We will test the hypothesis that neuronal inhibition by MrgX1 agonists involves G?i/o-dependent inhibitions of downstream high-voltage-activated (HVA) Ica and cAMP production. We will then examine if 179 and 891 potentiate the inhibition of HVA Ica by MrgX1 agonists in DRG neurons and in human neuronal HC-1 cells. Finally, we will determine whether PAMs also enhance MrgX1 inhibition of spinal nociceptive transmission. Aim 2 will provide important knowledge about neurophysiologic mechanisms for pain inhibition by MrgX1 agonists and PAMs. In Aim 3, we will examine the interaction between MrgX1/C and mu-opioid receptor (MOR) and its implication in morphine analgesia. In HEK293T cells, native DRG neurons, and HC-1 cells, we will test the hypothesis that MrgX1/C can interact physically with MOR. Furthermore, activation of MrgX1/C leads to co-internalization and sorting of MORs into the recycling pathway. We will examine if MrgX1 agonists enhance morphine inhibition of cAMP, potentiate morphine analgesia, and reduce morphine tolerance under neuropathic pain conditions. Aim 3 will uncover the physical and functional interplay between MrgX1/C and MOR, which may help improve morphine analgesia. Findings from the proposed studies will help us to conceptualize the biological basis of pain inhibition by MrgX1, and offer potential clinical translatability for the use of MrgX1 agonists and PAMs as novel pain therapies.